Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists

CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists.

All constructs were sequenced for correctness.
CD28 and CD86-eGFP (A206K): DNA coding for CD28 and CD86 was kindly provided by Davide Calebiro (University of Birmingham, UK) and was digested with Xhol and BamHI restriction enzymes and inserted into similar cut pEGFP-N1 vector.

Generation of CLEC-2 nanobodies
Nanobodies targeted against the extracellular domain of platelet receptor CLEC-2 were generated in collaboration with VIB Nanobody Core (Belgium). In short, immunisation of a llama occurred by subcutaneous injection of 100 µg recombinant human CLEC-2 (residues 55-229 with an N-terminal His6-tag) on days 0, 7, 14, 21, 28 and 35. Anti-coagulated blood was collected for lymphocyte preparation on day 40.
From the lymphocytes a VHH library was constructed consisting of more than 108 independent transformants, with 81% harbouring the right size insert in the phagemid vector (pMECS-GG). Three rounds of panning on solid-phase coated CLEC-2 antigen (100 µg/mL) were performed with enrichment for antigen-specific phages assessed after each round. Subsequently, 285 colonies were analysed by ELISA for the presence of CLEC-2 antigen specific nanobodies in crude periplasmic extracts. The screening revealed 107 positive colonies of which 48 represented unique nanobody sequences belonging to 12 different CDR3 groups. The library of clones was provided by VIB as E. coli TG1 harbouring phagemid pMECS-GG containing nanobody genes.

Production of CLEC-2 nanobodies in E. coli
CLEC-2 Nbs received in the pMECS-GG vector contain an N-terminal PelB signal sequence that targets the nanobody to the perisplasmic space of E. coli allowing their extraction from the periplasm. All 48 Nbs were expressed from small scale TG1 E. coli cultures for initial screening. Selected Nbs were then expressed from the WK6 E. coli strain (provided by VIB) as follows. WK6 colonies transformed with CLEC-2 Nb construct were grown overnight at 37°C in LB broth containing 100 µg/ml ampicillin.
Overnight cultures were used to inoculate 1L TB medium (2.3g/L KH2PO4, 16.4 g/L K2HPO4.3H2O, 12 g/L Tryptone, 24 g/L Yeast Extract, 4 mL/L Glycerol) 1:300 dilution supplemented with 100 µg/ml ampicillin. Cultures were incubated at 37°C, 180 rpm until an OD590 of 0.6-0.9 was reached. Nanobody expression was then induced with Nanobodies were purified from the periplasmic extract using nickel-coated beads affinity columns followed by size exclusion chromatography on a HiLoad 26/600 Superdex 75 pg column (Cytiva). The concentration of purified nanobody was determined via Nanodrop measuring absorbance at 280 nm according to the manufacturer's protocol. For his-tag cleavage of LUAS-2, thrombin (1 unit/ml) was added to LUAS-2-bound his-tag beads and incubated overnight at room temp. The following day, AEBSF (20 mg/ml stock) was added and incubated for 10 min at 4°C to inactivate residual thrombin.

Production of LUAS-2-Fc in mammalian cells
HEK293T cells were cultured at 37°C and 5% CO2 in complete DMEM (Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, 1% penicillin, 1% streptomycin and 1% glutamine). LUAS-2-Fc DNA was transiently transfected into mammalian HEK293T cells at 60% confluency using polyethylenimine (PEI Max MW 40,000, Polysciences) according to the manufacturer's instructions and expressed as secreted protein, due to the presence of an N-terminal secretion signal, into the culture media. LUAS-2-Fc secreted into the medium was collected after four days and subsequently purified using protein A affinity chromatography followed by size exclusion chromatography on a HiLoad 26/600 Superdex 75 pg column (Cytiva). The concentration of purified nanobody was determined via Nanodrop measuring absorbance at 280 nm according to the manufacturer's protocol.

CLEC-2 and podoplanin protein expression and purification
The cDNA sequences for the extracellular domain of human CLEC-2 (residues 55-229) and podoplanin were cloned into pHLSEC (Adegene) and pFUSE-Fc (InvivoGen) expression vectors respectively. Both constructs were expressed in house and sequenced for correctness. Recombinant human his6-CLEC-2 and recombinant human podoplanin-rFc were expressed in HEK293T cells by transient transfection using PEI (PEI Max MW 40,000) in serum free DMEM. After 4 days, proteins secreted into the medium were collected. Recombinant human his6-CLEC-2 and podoplanin-rFc were purified in a gravity purification column using Nickel-NTA resin (ThermoFisher) and protein A-coated beads (ThermoFisher) respectively. SDS-PAGE was used to confirm protein purity.

CRISPR-Cas9-mediated podoplanin knockout in HEK293T cells
Plasmid based CRISPR-Cas9 gene editing was used to knockout podoplanin expression in HEK293T cells. 2 Briefly, guide sequences targeting exon 1 of the human podoplanin gene were identified using the Sanger CRISPR Finder and selected on the least potential off-target binding. 3

Platelet lysis and protein phosphorylation
For Western blotting, whole platelet lysates (5x10 8 /ml) were prepared as reported. 4 Platelets were pre-treated with PRT-060318 (1 µM Figure 1A). Coverslips were acid cleaned with 1M HCl followed by 100% ethanol and air dried.

Single molecule photobleaching analysis and modelling
Fluorescent spots were localised using the ImageJ 5 plugin ThunderSTORM 6 on the average of the first 5 frames in the sequence, extracting the spots centre position and Gaussian width (σ). Spots with a σ below 50 nm and above 250 nm, or within a distance of 3.5 pixels (576 nm) of another spot, were excluded. Image sequences with aberrant imaging conditions were excluded based on the overall fluorescent decay.
Photobleaching traces were extracted from a ROI with a 2 pixel radius (329 nm). A ring-shaped ROI with an inner radius of 3.5 pixels (576 nm) and an outer radius of 5 pixels (832 nm) was used for background subtraction. Photobleaching analysis in quickpbsa was run with a step detection threshold of 20 counts and otherwise default parameters. However, to increase the robustness to photophysics the Bayesian refinement step in the quickpbsa analysis was not used, since this step relies on reproducible brightness of a single fluorophore and could be affected by blinking. This approach is valid for small fluorophore numbers (<10). 7 The eGFP number distributions obtained for CD86 and CD28 were modelled with binomial distributions using least-squares fitting from the optimised module of the python package scipy 8 (Supplementary Figure 3). For CD86, the model function is: (1) With the fraction of double spots rrc as a free parameter. For the monomers the labelling efficiency is set to 100%, since unlabelled monomers cannot be detected.
For CD28, we assume that CD28 is fully dimerised and that some CD28 dimers are randomly co-localised within one diffraction-limited volume, which would explain the observation of bright spots containing 3 or 4 eGFP copies. The model function is then given as the sum of two binomial probability distributions: with the fraction of double spots rrc and the labelling efficiency ple as free parameters.
The model yields a labelling efficiency of 58%. The labelling efficiency could be 8 underestimated due to blinking or unresolved steps in the analysis. The fraction of double spots in the model is 32%, which is consistent with the fraction of double spots observed in the measurement with the purely monomeric CD86 (24%).
The eGFP number distribution obtained for CLEC-2 was modelled with a binomial distribution using least squares fitting with python scipy. 8 For the mixture of monomers and dimers, the model function is: with the fraction of double spots rrc and the labelling efficiency ple as fixed parameters which were extracted from the modelling of CD28 data. The model yields a fraction of dimers of 64%.

Flow cytometry to measure CLEC-2 expression in cell lines
To

Flow cytometry to measure CLEC-2 expression in human platelets and humanised hCLEC-2 KI mouse platelets
To measure expression of human CLEC-2 on the surface of human and hCLEC-2 KI mouse platelets, the platelets were stained with AYP1 anti-hCLEC-2 antibody (66 nM) followed by anti-mouse Alexa Fluor-647 secondary antibody staining (1:400) at room temp. Control samples with no staining and secondary antibody staining only were also analysed. The samples were acquired (FL4) and analysed as described above.
Histograms were made in cSampler Software (BD Biosciences, USA).

CLEC-2 nanobody binding with flow cytometry
To investigate CLEC-2 nanobody binding with flow cytometry washed platelet samples were incubated with the nanobodies (7 nM) for 15 min at room temp followed by Alexa Fluor-647 anti-6-His tag antibody secondary labelling (5 µg/ml). Control samples with no staining and secondary antibody staining only were also analysed. The samples were acquired (FL4) and analysed as described above. Histograms were made in cSampler Software (BD Biosciences, USA).

Supplementary Figures
Supplementary Figure 1